Analytical procedures and compositions therefor

ABSTRACT

ANALYTICAL PROCEDURE FOR THE QUANTITATIVE DETERMINATION OF CREATININE IN BLOOD SERUM WHEREIN A NEUTRAL OR ALKALINE PH UP TO ABOUT 10.3, PREFERABLY ABOUT 9.5 TO ABOUT 10.3, IS DEVELOPED IN THE SAMPLE-REGENT BLANK PROCEDURE. A SIGNIFICANT ADVANTAGE OF THIS PH CONTROL TECHNIQUE IS THAT IT ELMINATES THE NEED FOR A PROTEIN-FREE FILTRATE.

United States Patent O 3,705,013 ANALYTICAL PROCEDURES AND COMPOSITIONS THEREFOR Peter B. Dewhurst, San Gabriel, Calif., assignor to Xerox Corporation, Rochester, N.Y. No Drawing. Filed Jan. 5, 1970, Ser. No. 800 Int. Cl. G01n 21/00, 33/16 US. Cl. 23-230 B 14 Claims ABSTRACT OF THE DISCLOSURE Analytical procedure for the quantitative determination of creatinine in blood serum wherein a neutral or alkaline pH up to about 10.3, preferably about 9.5 to about 10.3, is developed in the sample-reagent blank procedure. A significant advantage of this pH control technique is that it eliminates the need for a protein-free filtrate.

BACKGROUND OF THE INVENTION This application relates to an analytical procedure for the quantitative determination of creatinine in body fluids, particularly blood serum.

Creatinine is a waste product which is removed from the blood stream by the kidneys. Its normal concentration is 1 to 2 mg. percent. In the past, the quantitative determination of creatinine has been a most valuable tool in the diagnosis of urinary obstruction, intestinal obstruction, and nephritis, an inflammation of the kidney caused by infection, the degenerative process, or vascular disease. In cases of chronic nephritis, a high concentration of urea may be found with a normal concentration of creatinine. On the other hand, if the concentration of creatinine in the blood rises above 5 mg. percent, the prognosis is grave. In terminal stages of nephritis, values may rise as high as 35 mg. percent. However, in cases of urinary obstruction or acute nephritis in which there has not been any permanent renal injury, the creatinine value may become as high as to 30 mg. percent and return to normal when the pathological condition is relieved.

In 1886 Jaffe described the color reaction between creatinine and alkaline picrate. In 1914 Folin introduced this reaction for creatinine determination in blood, urine and tissues and, since that time, it has become, under the name of Jafles reaction, the most commonly used method for creatinine analysis in clinical chemistry.

Slot (Scandinav. J. Clin. Lab. Investigation, vol. 17, pages 381-387, 1965) presented a new J affe method based upon the observation that creatinine solutions and plasma filtrates behave differently when acidified after the picric color has been developed. Specifically, the Slot method is based upon the observation that the color produced by creatinine is less resistant to acidification than is the color produced by other Jaffe chromogens present in normal plasma. Slots method includes adding a picrate preparation to a protein-free filtrate, incubating for 90 minutes, and reading the color developed at 500 m After this first reading, a sulfuric acid preparation s added and a new reading taken 5 minutes later. The difference between the two readings is corrected for the loss of optical density shown by a reagent blank as a result of the acidification step which converts the alkaline picrate to picric acid.

Grafnetter et al (Clin. Chim. Acta. 17, pages 493-498, 1967) modified Slots technique by changing the ratio between the protein-free filtrate, the saturated picric acid solution and the sodium hydroxide solution from 8:1:1 to a 321:1 ratio thereby giving proper alkalinity for color develpment. Since Grafnetter et al. found that a pH below 4.0 resulted in turbidity, a small amount of glacial acetic acid was utilized in place of sulfuric acid to obtain "ice bulfering at the pH of the final reading (i.e., approximately 4.0). Finally, since they observed that maximum color development was reached in about 40 minutes with protein-free filtrates, they cut the Slot incubation period in half or to about 45 minutes.

As indicated above, both the original Slot method and the Grafnetter et al. modification thereof require a protein-free filtrate. This limits the overall utility of such procedures in automated determinations since it is necessary to either manually or automatically obtain such a protein-free filtrate. To do so, obviously, either requires operator time, a separate machine and/or an analyzer which automatically prepares protein-free filtrates. No matter which approach is taken, the effective time for running the creatinine determination is increased over the time necessary for a determination which does not require a protein-free filtrate. Accordingly, it would be desirable to have a creatinine determination techique which did not require a protein-free filtrate. That is, one which can utilize an unmodified blood sample so that the overall determination time is not increased by those steps necessary to obtain the protein-free filtrate.

In addition, the Slot technique requires correction for the optical density associated with the reagent blank when the alkaline picrate is converted to picric acid during the acidification step. His technique therefore requires four distinct optical density readings: 1) after incubation of the protein-free filtrate-alkaline picrate reagent reaction mixture; (2) after the acidification of the protein-free filtrate-picrate reagent reaction mixture; (3) the reagent blank; and (4) the acidified reagent blank. For high-speed automated analyzers, it would be desirable to limit the number of optical density readings to two without adversely affecting the accuracy and precision of the analytical data obtained thereby.

OBJECTS OF THE INVENTION It is therefore an object of the present invention to provide a novel method for the quantitative determination of creatinine in body fluids.

It is a further object of the present invention to provide an analytical procedure for the quantitative determination of creatinine in body fluids which does not require a protein-free filtrate.

It is a further object of the present invention to provide an analytical procedure for the quantitative determination of creatinine in blood serum which requires only two optical density readings.

It is a further object of the present invention to describe novel reagent compositions adapted for use in analytical procedures for the quantitative determination of creatinine in body fluids.

These and still further objects, features and advantages of the present invention will become apparent upon consideration of the following detailed disclosure of specific exemplary embodiments thereof.

BRIEF SUMMARY OF THE INVENTION The above and still further objects of the present inven tion are achieved, in accordance therewith, by adding an alkaline picrate preparation, adapted to develop a pH of about 12.3 and above, preferably 12.4, to an aliquot of blood serum, incubating the reaction mixture at 37 C. for about 14 to about 20 minutes, preferably about 14 to about 17 minutes, or until such time as there is maximum color development, and taking a single optical density measurement at 500 m Except for the shorter incubation period, this color development technique, wherein both creatinine and non-creatinine chromogens contribute to color development, corresponds to that described by Slot and Grafnetter et al. The improvement of this invention comprises concomitantly with the aforementioned procedure conducting a secondary procedure which establishes a reaction mixture pH Where (1) creatinine does not contribute to the optical absorbance of the solution and (2) non-creatinine chromogens offer substantially the same optical absorbance as they do in the primary highly alkaline test procedure. The pH of the reaction mixture developed in this secondary test should be neutral or alkaline up to a pH on the order of about 10.3, preferably about 9.5 to 10.3. After incubation for the same time period as the primary test, during which substantially only the color associated with the non-creatinine chromogens is developed, a single optical density measurement is taken at 500 m The difference between the two optical density measurements corresponds to the concentration of creatinine in the serum aliquot.

Since neither reaction mixture developed in the analytical procedure of the present invention is strongly acidified, it is not necessary to use a protein-free filtrate in the course of the determination. This is a significant advantage since the time, labor, etc. normally associated with the production of such a filtrate are saved while the final analytical result can be determined at lower cost in a shorter time period.

Avoiding the acidification technique offers another advantage. That is, since the secondary test is conducted at a neutral or alkaline pH, the alkaline picrate is not converted to picric acid and, accordingly, optical density measurements need not be taken to correct for the difference in optical absorbance caused by conversion of the alkaline picrate to picric acid. Thus, two of the optical density readings associated with the Slot technique can be eliminated without adversely affecting the accuracy of the overall determination.

The incubation period utilized in the analytical procedure of the present invention is, as indicated above, on the order of about 14 to about 20 minutes, preferably on the order of about 14 to about 17 minutes. After about 17 minutes, there is obtained color development attributable to non-creatinine chromogens which is not completely compensated for in the secondary test (i.e., the alkaline serum blank). However, it should be understood that longer incubation periods can be utilized, if desired, with a possible concomitant loss of accuracy and precision in the final analytical result.

As indicated above, the presently preferred pH range for conducting the secondary test is between about 9.5 and about 10.3. At the lower limit, there will be minimum color development attributable to the creatinine in the sample aliquot. The optical absorbance of the resultant solution, however, will not be as great as the optical absorbance of such a solution having a pH of around 10.3. This will result in somewhat less than complete compensation for all the non-creatinine chromogens in the sample aliquot.

At the upper value (i.e., about 10.3 however, the optical absorbance of the non-creatinine chromogens will more closely approximate the optical absorbance of such materials at the higher pH associated with the test procedure. There will also be some minor color development associated with creatinine at thi pH (i.e., about 10.3). This minor color contribution from creatinine in combination with the color contribution attributable to the noncreatinine chromogens more closely approximates the total color development associated with the non-creatinine chromogens in the test procedure, such that the difference in optical density readings between the test procedure and the alkaline serum blank procedure, at this pH value, is an accurate indicator of the concentration of creatinine in the sample material. Thus, within the preferred range, pH values at the upper end thereof on the order of about 10.0 to about 10.3, represent the optimum pH values for obtaining analytical data from which the concentration of creatinine can best be determined.

The reagents can be added in either solid or liquid form. Liquid reagents, however, have a greater propensity towards chemical reaction and are generally known to be more sensitive to light and other portions of the electromagnetic spectrum. Accordingly, it is preferred to store and add the reagents in solid form whenever possible. When added in the solid state, the reagents can be in powdered or tabletted form, either singly or in combination with other compatible reagents. A disadvantage of storing two or more powdered reagents together is the extreme amount of surface area available for chemical reaction during the prolonged storage periods. Because of the ease of handling and the prolonged storage which can be obtained therewith, reagent tablets are the presently preferred form of adding the reagent materials, though it should be clearly understood that the actual form of the materials is not an essential, or critical, feature of the present invention.

In the reagent formulations specifically identified in the examples below, other alkali picrates, such as lithium picrate, can be substituted for the sodium picrate; and other buffers, or combination of buffers, can be substituted for the sodium carbonate and the sodium sulfate, provided the appropriate pH is developed in the resultant solution. The alkaline picrate and alkalizing solutions can be formulated as a single test solution. It would then, however, be necessary to provide a corresponding sodium picrate solution to be used solely in the blank reaction and which develops the alkaline pH taught herein. The amounts of reagents can be varied and certain tabletting additives, such as dextran, PEG-6000 and Dupanol can be eliminated, or substituted for by other tabletting additives. The use of Tergitol NP-44, however, has been found desirable since it assists in the maintenance of a clear solution necessary for optical density measurements.

DESCRIPTION OF SPECIFIC EMBODIMENTS The following examples are given to enable those skilled in the art to more clearly understand and practice the invention. They should not be considered as a limitation upon the scope of the invention but merely as being illustrative thereof.

The reagent formulations utilized in Examples I-XVIII are prepared as follows:

(a) Sodium picrate reagent: dissolve the following weights of materials in distilled water:

Mg. Sodium picrate 640 Na SO 4170 NagCO Dextran 80 Tergitol N P-44 PEG-6000 100 Dupanol 40 After thorough mixing, the solution is made up to 100 ml. with distilled water.

(b) Alkalizing reagent: dissolve the following weights of materials in distilled water:

Mg. LiOH 240 KCl 1200 Na SO 3440 Dextran PEG-6000 120 the solution is made up to 100 ml.

bated for 5 minutes at 37 C. 200 ,ul. of the same serum or standard as used in the test reaction is added and incu- C. for 15 minutes. The pH of this solution bated at 37 is about 100-103. After the 15 minute incubation period, the optical density of the resultant solution is measured at 00 mp.

A standard curve (plotting net optical density vs. mg. percent creatinine) is prepared using solutions having known concentrations of creatinine therein. To determine the creatinine concentration in a particular serum aliquot, the net optical density (defined as the dilference between the test reaction and the blank reaction optical density measurements) is determined, and reference is made to the standard curve whereby optical density units are converted to mg. percent creatinine.

EXAMPLES I-X Ten randomly-selected serum samples were analyzed for their creatinine concentration in accordance with the procedure described above. Concurrently, the results of these analytical determinations were compared against corresponding values obtained using the method described by Kingsley and Shaifert, Standard Methods of Clinical Chemistry, vol. 1, pages 55-59 (1953), one of the presently recognized standards for this type of determination. The results of these determinations, in mg. percent creatinine, are given below in Table I:

TABLE I Reference Procedure procedure of described Kingsley herein (mg. dz Shafiert percent (mg. percent creatinine) creatinine) Though some minor variations are evident from Table I, a graph of this data would indicate that there is essentially a 1:1 correlation between the procedure of the present invention and the reference procedure of Kingsley and Shatfert with the correlation line going through the zero intercept. This means that the data obtained with the procedure described herein has good correlation with a presently recognized standard procedure for creatinine determinations. Furthermore, it indicates that the data obtained with the procedure described herein may be used as determined and need not be modified by certain factors to place it in a form which has meaning to the physician. This latter result follows from the 1:1 correlation and the fact that the correlation line passes through the zero intercept.

EXAMPLES XI-XVIII The test, blank and reference procedures of Examples I-X are repeated with 8 diiferent, randomly-selected serum samples. The results of these determinations are given below in Table II:

Once again, though minor variations are evident from the data presented in Table H, there is essentially a 1:1

correlation between the procedure of the present invention and the reference procedure of Kingsley and Shaifert. As the correlation line passes very close to the zero intercept, the data is directly usable and need not be modified by certain factors to place it in a form understandable by the physician.

EXAMPLES XIX-XXVIII The reagent formulations utilized in Examples XIX- XXVIII are as follows:

(a) Sodium picrate tablet:

In the test reaction, one sodium picrate tablet and one alkalizing tablet are dissolved in 0.500 ml. of distilled water and incubated for 5 minutes at 37 C. ,ul. of serum or standard is added and incubated at 37 C. for 15 minutes. The optical density is then read at 500 m In the blank reaction, one sodium picrate tablet is dissolved in 0.500 ml. of distilled water and incubated for 5 minutes at 37 C. 80 l. of the same standard or serum sample as used in the test reaction is added and incubated at 37 C. for 15 minutes. The optical density is then read at 500 m As set forth above, a standard curve is derived using solutions having known concentrations of creatinine therein. From the difference in the optical density measurements between the test and blank procedures (i.e., the net optical density), the mg. percent creatinine in the serum sample is determined from the standard curve.

As in the preceding examples, the combination of the sodium picrate tablet, the alkalizing tablet and the serum sample develops a solution pH of about 12.35 or above. In the blank procedure, the combination of the sodium picrate tablet with the diluted serum aliquot develops a solution pH on the order of about 10.0-10.3.

Ten different, randomly-selected serum samples were analyzed for their creatinine concentration and compared against corresponding values obtained using the Kingsley and Shaffert reference procedure identified above. The results for these determinations are given below in Table III:

With the reference procedure values being plotted on the x-axis, the slope of the correlation line is 1.02:1 with a y-axis intercept equal to about 0.04. This represents good correlation, well within permissible clinical chemistry variations, between the procedure of the present invention and the accepted procedure of Kingsley and Shatfert. The slight deviation from the zero intercept corresponds to less than 0.1 mg. percent creatinine and, though not desirable, is within permissible limits for this determination.

EXAMPLES XXIX-XXXVI To a standard solution containing 20 mg. percent creatinine, there is added lithium hydroxide and sufiicient sodium picrate to react with all creatinine present therein. The pH of the resultant solution is at least 12.35. Equal aliquots of this solution are pipetted into 8 separate tubes and a small amount of acid, in increasing increments, is added to the succession of tubes. All volumes are made equal by the addition of small amounts of distilled water. The tubes are incubated for minutes at 37 C. whereafter the optical density and the pH of each solution are determined. The optical density measurements are corrected by subtracting therefrom the optical density associated with sample-reagent blanks prepared under identical conditions, such as incubation, pH, etc. The net optical density (i.e., the difference in optical density between the test procedure and reagent blank procedure) versus the pH of the resultant solution is given below in Table IV:

The data presented in Table IV illustrates that there is essentially no color development between creatinine and the alkaline picrate at a solution pH less than about 9.55. Accordingly, in the Slot and Grafnetter et a1. procedures it is unnecessary to acidify the resultant solution to a pH on the order of about 4.0 or below. Rather, the pH of the blank reaction is made somewhat less alkaline, but not acidic, as described herein, wherefore the correction for the conversion of alkaline picrate to picric acid is no longer needed.

At this point, it should be re-emphasized that the analytical data obtained with the procedure of the present invention, which has been shown to be in good correlation with corresponding values obtained with the Kingsley and Shatfert reference procedure, have been obtained without the use of a protein-free filtrate. This represents a significant advantage since the labor, time, cost, etc. normally associated with producing such a protein-free filtrate are now saved and the quantitative determination can, accordingly, be conducted at lower cost in a shorter time period.

While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the invention. For example, the optical density measurements can be taken at other wavelengths, such as 490 to 520 m;;.. In addition, many modifications may be made to adapt a particular situation, material or formulation, to

the spirit of the present invention without departing from its essential teachings.

What is claimed is:

1. A blank determination process for use in conjunction with the quantitative determination of creatinine cornprising mixing an aliquot of proteinaceous body fluid sample material with an alkali picrate and a buffer adapted to establish a solution pH at which creatinine does not contribute significantly to the optical absorbance thereof and non-creatine chromogens offer substantially the same optical absorbance as said non-creatinine chromogens offer at a pH of about 12.3 and above, incubating the resultant solution, and thereafter determining the optical absorbance thereof.

2. The process of claim 1 wherein said sample material comprises blood serum which has not been subjected to conditions which would cause the precipitation or removal of proteinaceous components therefrom.

3. The process of claim 1 wherein said resultant solution is incubated for about 14 to about 20 minutes at 37 C.

4. The process of claim 1 wherein said resultant solution is incubated for about 14 to about 17 minutes at 37 C.

5. The process of claim 1 wherein the optical absorbance of the incubated solution is measured at about 500 m 6. A process for the measurement of the optical absorbance of the body fluid sample aliquot-alkaline picrate reagent blank, said measurement being useful in a procedure for the quantitative determination of creatinine, said process comprising mixing an aliquot of proteinaceous body fluid sample material with an alkali picrate and a buffer adapted to develop a neutral or alkaline pH up to about 10.3, incubating the resultant solution, and thereafter determining the optical absorbance thereof.

7. The process of claim 6 wherein said sample material comprises blood serum which has not been subjected to conditions which would cause the precipitation or removal of proteinaceous components therefrom.

8. A process for the measurement of the optical absorbance of a body fluid sample aliquot-alkaline picrate reagent blank, said measurement being useful in a procedure for the quantitative determination of creatinine, said process comprising mixing a proteinaceous aliquot of blood serum with an alkali picrate and a buffer adapted to develop a solution pH of about 9.5 to about 10.3, incubating the resultant solution, and thereafter measuring the optical absorbance thereof.

9. The process of claim 8 wherein said buffer is adapted to produce a solution pH on the order of about 10.0 to about 10.3.

10. A process for the quantitative determination of creatinine comprising providing a first vessel for conducting a test procedure therein, providing a second vessel for conducting a blank procedure therein, mixing in said first vessel an aliquot of proteinaceous body fluid sample material with an alkali picrate and a buffer adapted to develop a pH of at least about 12.3, mixing in said second vessel an aliquot of proteinaceous body fluid sample material with an alkali picrate and a buffer adapted to develop a solution pH at which creatinine does not contribute significantly to the optical absorbance of the resultant solution and noncreatinine chromogens offer substantially the same optical absorbance as said noncreatinine chromogens offer at the alkaline pH developed in said first vessel, incubating the reaction mixture in each vessel a sutficient period of time to obtain appropriate color development, measuring the optical absorbance of the reaction mixture in each vessel, and substracting the optical absorbance of the reaction mixture in said second vessel from the optical absorbance of the reaction mixture in said first vessel to yield a net optical absorbance difference which is correlated to the concentration of creatinine in said sample material.

10 11. The process of claim 10 wherein the reaction mix- References Cited gilljrguinzgaggufizzel is incubated at 37 C. for about 14 to UNITED STATES PATENTS 12. The process of claim 10 wherein the optical absorb- 3,557,018 1/ 1971 Scheuerbrandt 23-230 B ance of each reaction mixture is measured at about 5 3,560,161 2/1971 Webb R X 500 I OTHER REFERENCES 13. The process of claim 10 wherein the buffer associated with said second vessel is adapted to produce 21 Clark et Anal Chem October 1949 solution pH on the order of about 9.5 to about 10.3. 12184221 14. The process of claim 10 wherein the buifer associ- 10 ated with said second vessel is adapted to produce a MORRIS WOLK Primary Exammer solution pH on the order of about 10.0 to about 10.3. S s is a t Examiner 

